Quinacridone derivatives as labelling reagents for fluorescence detection of biological materials

ABSTRACT

Disclosed are new quinacridone dye derivatives having characteristic fluorescence lifetimes. Also disclosed are methods for labelling target biological materials employing the quinacridone dyes and use of the labelled materials in biological assays. The quinacridone derivatives have the following structure: 
                         
in which Z 1  and Z 2  independently represent the atoms necessary to complete one ring, two fused ring, or three fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; R 3 , R 4 , R 5 , R 6 , R 7  and R 8  are independently selected from hydrogen, halogen, amide, hydroxyl, cyano, nitro, mono- or di-nitro-substituted benzyl, amino, mono- or di-C 1 -C 4  alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C 1 -C 6  alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C 1 -C 20  alkyl, aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group -E-F and the group —(CH 2 —) n Y; R 1  and R 2  are independently selected from hydrogen, mono- or di-nitro-substituted benzyl, C 1 -C 20  alkyl, aralkyl, the group -E-F and the group —(CH 2 —) n Y; E is a spacer group, F is a target bonding group; Y is selected from sulphonate, sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl; and n is an integer from 1 to 6.
 
     The invention also relates to a set of different fluorescent quinacridone dye derivatives, each dye having a different fluorescence lifetime, the set of dyes being particularly useful for multiparameter analysis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a filing under 35 U.S.C. § 371 and claims priorityto international patent application number PCT/GB02/02537 filed May 30,2002, published on Dec. 12, 2002 as WO02/099432, and to foreignapplication number 0113434.5 filed in Great Britain on Jun. 4, 2001, theentire disclosures of which are hereby incorporated by reference.

The present invention relates to new fluorescent labels. In particularthe invention relates to new quinacridone derivatives that can be usedas labels for attachment to target biological materials. The inventionalso relates to methods for labelling target biological materials anduse of such labelled materials in biological assays.

There is an increasing interest in, and demand for, fluorescent labelsfor use in the labelling and detection of biological materials.Fluorescent labels are generally stable, sensitive and a wide range ofmethods are now available for labelling biomolecules. Typically, theemission spectrum of a fluorescent dye is a characteristic property ofthe is dye. Measurements of the fluorescence intensity, the fluorescencelifetime, or fluorescence polarisation may be used in the detection andquantitation of materials labelled with that dye. One problem withmeasurements of fluorescence intensity as a means of detecting and/ormeasuring the concentration of a fluorescent labelled biomolecule isthat background fluorescence may interfere with the measurement. Thus,in order to obtain improvements in the sensitivity of fluorescencedetection, it is highly desirable to improve the signal-to-noise ratio.

One means of overcoming the problem of background noise has been throughthe use of long wavelength dyes, for example, the cyanine dyes Cy™5 andCy7, as disclosed in U.S. Pat. No. 5,268,486 (Waggoner et al). Thesedyes emit in the 600-750 nm region of the spectrum, where backgroundfluorescence is much less of a problem. Another means of improving thesignal-to-noise ratio in fluorescence measurements is through the use oftime-resolved fluorescence, for example by using fluorescent labelsbased on lanthanide chelates, eg. Eu³⁺ and Tb³⁺ (Selvin et al, U.S. Pat.No. 562,282). In time-resolved fluorescent labels, the fluorescenceemission is typically longer than that of the background fluorescence,which may therefore be gated out using appropriate instrumentation.

Linear trans-quinacridones are highly fluorescent and quinacridonederivatives have been developed as organic pigments (U.S. Pat. No.2,844,484 (Reidinger, A. D. et al), U.S. Pat. No. 3,386,843 (Jaffe, E.E. et al)), for use in high sensitivity photosensors and organiclight-emitting diodes and optical probes. Liu, P-H et al(J.Photochem.Photobiol., (2000), 137, 99-104) have synthesised a numberof 5,12-N,N′-dialkyl-2,9-dialkoxy quinacridones and have investigatedtheir spectral properties. Klein, G. et al (J.Chem.Soc.Chem.Commun.,(2001), 561-2) have prepared ethylenediamine functionalized quinacridonederivatives for use as fluorescent metal sensors.

Val'kova, G. et al (Dokl. Akad. Nauk. SSR, (1978), 240(4), 884-7) havemeasured the fluorescence lifetime of quinacridone, however, to date,there appear to be no reports relating to the use of quinacridones asdyes suitable for labelling and the detection of biological materialssuch as nucleic acids, peptides, proteins, antibodies, drugs, hormones,cells and the like. The present invention therefore describesmodifications of the quinacridone chromophore, to produce a range ofquinacridone derivatives which are useful for labelling biologicalmaterials. The quinacridone derivatives of the present inventionmoreover provide a valuable set of fluorescent labels having a commoncore structure and which are particularly useful for multiparameteranalysis. In each dye of a set of dyes, the absorption and emissionspectra remain essentially the same, whilst the fluorescence lifetimesof the dyes vary. Thus, it is possible to use a common excitation sourceand determine the fluorescence lifetimes at the same emissionwavelength, thereby simplifying requirements for detectioninstrumentation used in multiparameter experiments. Another advantage ofthe dyes according to the present invention is that the fluorescenceemission wavelengths and lifetimes of the quinacridone derivatives aregenerally longer than the lifetimes of other fluorescent labels as wellas naturally occurring fluorescent materials, such as proteins andpolynucleotides, thereby allowing easy discrimination from backgroundfluorescence in assays utilising such dyes.

Accordingly in a first aspect, the present invention provides use of areagent for labelling a target biological material, wherein said reagentis a dye of formula (I):

wherein:

-   groups R³ and R⁴ are attached to the Z¹ ring structure and groups R⁵    and R⁶ are attached to the Z² ring structure;-   Z¹ and Z² independently represent the atoms necessary to complete    one ring, two fused ring, or three fused ring aromatic or    heteroaromatic systems, each ring having five or six atoms selected    from carbon atoms and optionally no more than two atoms selected    from oxygen, nitrogen and sulphur;-   R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected from hydrogen,    halogen, amide, hydroxyl, cyano, nitro, mono- or    di-nitro-substituted benzyl, amino, mono- or di-C₁-C₄    alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆    alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl,    aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group    -E-F and the group —(CH₂—)_(n)Y;-   R¹ and R² are independently selected from hydrogen, mono- or    di-nitro-substituted benzyl, C₁-C₂₀ alkyl, aralkyl, the group -E-F    and the group —(CH₂—)_(n)Y;-   E is a spacer group having a chain from 1-60 atoms selected from the    group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus    atoms and F is a target bonding group;-   Y is selected from sulphonate, sulphate, phosphonate, phosphate,    quaternary ammonium and carboxyl; and n is an integer from 1 to 6.

In a first embodiment of the first aspect, the dye of formula (I) is afluorescent dye wherein:

-   groups R³ and R⁴ are attached to atoms of the Z¹ ring structure and    groups R⁵ and R⁶ are attached to atoms of the Z² ring structure,    where Z¹ and Z² are hereinbefore defined;-   R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected from hydrogen,    halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄    alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆    alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl,    aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group    -E-F and the group —(CH₂—)_(n)Y; and-   R¹ and R² are independently selected from hydrogen, C₁-C₂₀ alkyl,    aralkyl, the group -E-F and the group —(CH₂—)_(n)Y;-   wherein E, F, Y and n are hereinbefore defined.

The quinacridone dyes according to the first embodiment of the firstaspect are particularly suitable for use as fluorescence lifetime dyes.In the context of the present invention, the term lifetime dye isintended to mean a dye having a measurable fluorescence lifetime,defined as the average amount of time that the dye remains in itsexcited state following excitation (Lakowicz, J. R., Principles ofFluorescence Spectroscopy, Kluwer Academic/Plenum Publishers, New York,(1999)). Alternatively, the dyes may be used in assays utilisingfluorescence polarisation.

Suitably, the fluorescent dyes according to the first embodiment of thefirst aspect exhibit a fluorescence lifetime in the range from 1 to 30nanoseconds. Preferably, the fluorescent lifetimes of the dyes are inthe range from 10 to 25 nanoseconds.

In a second embodiment of the first aspect, the dye of formula (I) is anon-fluorescent or substantially non-fluorescent dye wherein:

-   groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² are hereinbefore    defined; and wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶,    R⁷ and R⁸ comprises at least one nitro group.

In this embodiment, suitably, the at least one nitro group may beattached directly to the Z¹ and/or Z² ring structures. In thealternative, a mono- or di-nitro-substituted benzyl group may beattached to the R¹, R², R³, R⁴, R⁵, R⁶, R⁷ or R⁸ positions, whichoptionally may be further substituted with one or more nitro groupsattached directly to the Z¹ and/or Z² ring structures.

Preferably, in the first and second embodiments, at least one of groupsR¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ in the dye of formula (I) is the group-E-F where E and F are hereinbefore defined.

Suitably, the target bonding group F is a reactive or functional group.A reactive group of a dye of formula (I) can react under suitableconditions with a functional group of a target material; a functionalgroup of a dye of formula (I) can react under suitable conditions with areactive group of the target material such that the target materialbecomes labelled with the compound.

Preferably, when F is a reactive group, it is selected from succinimidylester, sulpho-succinimidyl ester, isothiocyanate, maleimide,haloacetamide, acid halide, vinylsulphone, dichlorotriazine,carbodiimide, hydrazide and phosphoramidite. Preferably, when F is afunctional group, it is selected from hydroxy, amino, sulphydryl,imidazole, carbonyl including aldehyde and ketone, phosphate andthiophosphate. By virtue of these reactive and functional groups the dyeof formula (I) may react with and covalently bond to target materials.

Suitably, Z¹ and Z² may be selected from the group consisting of phenyl,pyridinyl, naphthyl, anthranyl, indenyl, fluorenyl, quinolinyl, indolyl,benzothiophenyl, benzofuranyl and benzimidazolyl moieties. Additionalone, two fused, or three fused ring structures will be readily apparentto the skilled person. Preferred Z¹ and Z² are selected from the groupconsisting of phenyl, pyridinyl, naphthyl, quinolinyl and indolylmoieties. Particularly preferred Z¹ and Z² are phenyl and naphthylmoieties.

Preferably, at least one of the groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸of the dyes of formula (I) is a water solubilising group for conferringa hydrophilic characteristic to the compound. Solubilising groups, forexample, sulphonate, sulphonic acid and quaternary ammonium, may beattached directly to the aromatic ring structures Z¹ and/or Z² of thecompound of formula (I). Alternatively, solubilising groups may beattached by means of a C₁ to C₆ alkyl linker chain to said aromatic ringstructures and may be selected from the group —(CH₂—)_(n)Y where Y isselected from sulphonate, sulphate, phosphonate, phosphate, quaternaryammonium and carboxyl; and n is an integer from 1 to 6. Alternativesolubilising groups may be carbohydrate residues, for example,monosaccharides. Examples of water solubilising constituents includeC₁-C₆ alkyl sulphonates, such as —(CH₂)₃—SO₃— and —(CH₂)₄—SO₃ ^(—).However, one or more sulphonate or sulphonic acid groups attacheddirectly to the aromatic ring structures of a dye of formula (I) areparticularly preferred. Water solubility may be advantageous whenlabelling proteins.

Suitable spacer groups E may contain 1-60 chain atoms selected from thegroup consisting of carbon, nitrogen, oxygen, sulphur and phosphorus.For example the spacer group may be:

-   —(CHR′)_(p)—-   —{(CHR′)_(q)—O—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)—    where R′ is hydrogen, C₁-C₄ alkyl or aryl, which may be optionally    substituted with sulphonate, Ar is phenylene, optionally substituted    with sulphonate, p is 1-20, preferably 1-10, q is 0-10, r is 1-10    and s is 1-5.

Specific examples of reactive groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸and the groups with which R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ can reactare provided in Table 1. In the alternative, groups R¹, R², R³, R⁴, R⁵,R⁶, R⁷ and R⁸ may be the functional groups of Table 1 which would reactwith the reactive groups of a target material.

TABLE 1 Possible Reactive Substituents and Sites Reactive TherewithReactive Groups Functional Groups succinimidyl esters primary amino,secondary amino isothiocyanates amino groups haloacetamides, maleimidessulphydryl, imidazole, hydroxyl, amine acid halides amino groupsanhydrides primary amino, secondary amino, hydroxyl hydrazides,aldehydes, ketones vinylsulphones amino groups dichlorotriazines aminogroups carbodiimides carboxyl groups phosphoramidites hydroxyl groups

Preferred reactive groups which are especially useful for labellingtarget materials with available amino and hydroxyl functional groupsinclude:

where n is 0 or an integer from 1-10.

Aryl is an aromatic substituent containing one or two fused aromaticrings containing 6 to 10 carbon atoms, for example phenyl or naphthyl,the aryl being optionally and independently substituted by one or moresubstituents, for example halogen, hydroxyl, straight or branched chainalkyl groups containing 1 to 10 carbon atoms, aralkyl and C₁-C₆ alkoxy,for example methoxy, ethoxy, propoxy and n-butoxy.

Heteroaryl is a mono- or bicyclic 5 to 10 membered aromatic ring systemcontaining at least one and no more than 3 heteroatoms which may beselected from N, O, and S and is optionally and independentlysubstituted by one or more substituents, for example halogen, hydroxyl,straight or branched chain alkyl groups containing 1 to 10 carbon atoms,aralkyl and C₁-C₆ alkoxy, for example methoxy, ethoxy, propoxy andn-butoxy.

Aralkyl is a C₁ to C₆ alkyl group substituted by an aryl or heteroarylgroup.

Halogen and halo groups are selected from fluorine, chlorine, bromineand iodine.

Exemplary dyes according to the first embodiment of the first aspect areas follows:

-   i)    6-{2,9-dimethoxy-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoic    acid, diethyl ester;-   ii)    6-{2,9-dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoic    acid;-   iii)    6-{12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoic    acid.

The fluorescent dyes according to the present invention may be used tolabel and thereby impart fluorescent properties to a variety of targetbiological materials. Thus, in a second aspect, there is provided amethod for labelling a target biological material the method comprising:

-   i) adding to a liquid containing said target biological material a    dye of formula (I):

wherein:

-   groups R³ and R⁴ are attached to the Z¹ ring structure and groups R⁵    and R⁶ are attached to the Z² ring structure, where Z¹ and Z² are    hereinbefore defined;-   R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected from hydrogen,    halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄    alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆    alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl,    aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group    -E-F and the group —(CH₂—)_(n)Y;-   R¹ and R² are independently selected from hydrogen, C₁-C₂₀ alkyl,    aralkyl, the group -E-F and the group —(CH₂—)_(n)Y;-   where E, F, Y and n are hereinbefore defined; and-   ii) incubating said dye with said target biological material under    conditions suitable for labelling said target.

Suitably, the fluorescent dyes of the present invention wherein at leastone of the groups R¹ to R⁸ contains a charge, for example, quaternaryamino, may be used to bind non-covalently to charged biologicalmolecules such as, for example, DNA and RNA. Alternatively, fluorescentdyes of the present invention wherein at least one of the groups R¹ toR⁸ is an uncharged group, for example, a long chain alkyl, an arylgroup, or an ester group may be used to bind to and thereby labeluncharged biological molecules such as, for example, biological lipids,as well as to intact cell membranes, membrane fragments and cells.

In a preferred embodiment according to the second aspect, at least oneof groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ in the dye of formula (I) isthe group -E-F where E and F are hereinbefore defined. In thisembodiment, the fluorescent dyes may be used to covalently label atarget biological material. The target bonding group may be a reactivegroup for reacting with a functional group of the target material.Alternatively, the target bonding group may be a functional group forreacting with a reactive group on the target biological material. Themethod comprises incubating the target biological material with anamount of the dye according to the invention under conditions to form acovalent linkage between the target material and the dye. The target maybe incubated with an amount of a compound according to the presentinvention having at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ andR⁸ that includes a reactive or functional group that can covalently bindwith the functional or reactive group of the target biological material.

Suitable target biological materials include, but are not limited to thegroup consisting of antibody, lipid, protein, peptide, carbohydrate,nucleotides which contain or are derivatized to contain one or more ofan amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate andthiophosphate groups, and oxy or deoxy polynucleic acids which containor are derivatized to contain one or more of an amino, sulphydryl,carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups,microbial materials, drugs, hormones, cells, cell membranes and toxins.

Fluorescent dyes according to the present invention may be used in assaymethods that employ fluorescent labels for the detection and/ormeasurement of analytes using, for example, fluorescence intensity,fluorescence lifetime, or fluorescence polarisation measurements.Examples of such assays include protein-protein binding assays,immunoassays and nucleic acid hybridisation assays.

In a third aspect, there is provided a method for the assay of ananalyte in a sample which method comprises:

-   i) contacting the analyte with a specific binding partner for said    analyte under conditions suitable to cause the binding of at least a    portion of said analyte to said specific binding partner to form a    complex and wherein one of said analyte and said specific binding    partner is labelled with a fluorescent dye of formula (I):

wherein:

-   groups R³ and R⁴ are attached to atoms of the Z¹ ring structure and    groups R⁵ and R⁶ are attached to atoms of the Z² ring structure,    where Z¹ and Z² are hereinbefore defined;-   at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is the    group -E-F where E is a spacer group having a chain from 1-60 atoms    selected from the group consisting of carbon, nitrogen, oxygen,    sulphur and phosphorus atoms and F is a target bonding group;-   when any of said groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is not said group    -E-F, said remaining groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are    independently selected from hydrogen, halogen, amide, hydroxyl,    cyano, amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl,    carbonyl, carboxyl, C₁-C₈ alkoxy, acrylate, vinyl, styryl, aryl,    heteroaryl, C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid,    quaternary ammonium and the group —(CH₂—)_(n)Y; and,-   when any of groups R¹ and R² is not said group -E-F, said remaining    groups R¹ and R² are independently selected from hydrogen, C₁-C₂₀    alkyl, aralkyl and the group —(CH₂—)_(n)Y;-   wherein Y and n are hereinbefore defined;-   ii) measuring the emitted fluorescence of the labelled complex; and-   iii) correlating the emitted fluorescence with the presence or the    amount of said analyte in said sample.

Suitably, step ii) may be performed by measurement of the fluorescenceintensity, fluorescence lifetime, or fluorescence polarisation of thelabelled complex. Preferably, the measuring step ii) is performed bymeasuring the fluorescence lifetime, or the fluorescence polarisation ofthe labelled complex.

In one embodiment, the assay method is a direct assay for themeasurement of an analyte in a sample. A known or putative inhibitorcompound may be optionally included in the reaction mixture.

In a second, or alternative embodiment, the assay may be a competitiveassay wherein a sample containing an analyte competes with a fluorescenttracer for a limited number of binding sites on a binding partner thatis capable of specifically binding the analyte and the tracer. Suitably,the tracer is a labelled analyte or a labelled analyte analogue, inwhich the label is a fluorescent dye of formula (I). Increasing amounts(or concentrations) of the analyte in the sample will reduce the amountof the fluorescent labelled analyte or fluorescent labelled analyteanalogue that is bound to the specific binding partner. The fluorescencesignal is measured and the concentration of analyte may be obtained byinterpolation from a standard curve.

In a further embodiment, the binding assay may employ a two-step format,wherein a first component (which may be optionally coupled to aninsoluble support) is bound to a second component to form a specificbinding complex, which is bound in turn to a third component. In thisformat, the third component is capable of specifically binding to eitherthe second component, or to the specific binding complex. Either of thesecond or the third component may be labelled with a fluorescent dyeaccording to the present invention. Examples include “sandwich” assays,in which one component of a specific binding pair, such as a firstantibody, is coated onto a surface, such as the wells of a multiwellplate. Following the binding of an antigen to the first antibody, afluorescent labelled second antibody is added to the assay mix, so as tobind with the antigen-first antibody complex. The fluorescence signal ismeasured and the concentration of antigen may be obtained byinterpolation from a standard curve.

In particularly preferred embodiments, the measurement step may beperformed using fluorescence polarisation. Thus, when the fluorescenttracer is not bound to the specific binding partner, it will tumble andreorientate rapidly relative to the fluorescence lifetime of thefluorescent dye. When bound to the specific binding partner, the tracerwill tumble and reorientate slowly relative to the fluorescence lifetimeof the dye. The degree of polarisation is therefore proportional to theextent of binding of the fluorescent tracer in the sample and inverselyproportional to the amount of analyte in the sample.

Examples of analyte-specific binding partner pairs include, but are notrestricted to, antibodies/antigens, lectins/glycoproteins,biotin/streptavidin, hormone/receptor, enzyme/substrate or co-factor,DNA/DNA, DNA/RNA and DNA/binding protein. It is to be understood thatany molecules which possess a specific binding affinity for each othermay be employed, so that the fluorescent dyes of the present inventionmay be used for labelling one component of a specific binding pair,which in turn may be used in the detection of binding to the othercomponent.

The dyes according to the present invention may also be used in enzymeassays, utilising fluorescence polarisation measurements. An assay forthe detection of enzyme activity may be configured as follows. Areaction mixture is prepared by combining the enzyme and a fluorogenicsubstrate labelled with a fluorescent dye according to the presentinvention. A known or putative inhibitor compound may be optionallyincluded in the reaction mixture. The progress of the reaction may bemonitored by observing a change in fluorescence polarisation of thesample.

Thus, in a fourth aspect, there is provided an assay method for thedetermination of an enzyme in a sample, the method comprising:

-   i) providing a substrate for the enzyme wherein the substrate is    labelled with a fluorescent dye of formula (I):

wherein:

-   groups R³ and R⁴ are attached to atoms of the Z¹ ring structure and    groups R⁵ and R⁶ are attached to atoms of the Z² ring structure,    where Z¹ and Z² are hereinbefore defined;-   at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is the    group -E-F where E is a spacer group having a chain from 1-60 atoms    selected from the group consisting of carbon, nitrogen, oxygen,    sulphur and phosphorus atoms and F is a target bonding group;-   when any of said groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is not said group    -E-F, said remaining groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are    independently selected from hydrogen, halogen, amide, hydroxyl,    cyano, amino, mono- or di-C₁-C₄ alkyl-substituted amino, sulphydryl,    carbonyl, carboxyl, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl,    heteroaryl, C₁-C₂₀ alkyl, aralkyl, sulphonate, sulphonic acid,    quaternary ammonium and the group —(CH₂—)_(n)Y; and,-   when any of groups R¹ and R² is not said group -E-F, said remaining    groups R¹ and R² are independently selected from hydrogen, C₁-C₂₀    alkyl, aralkyl and the group —(CH₂—)_(n)Y;-   wherein Y and n are hereinbefore defined;-   ii) combining the labelled substrate with the enzyme under    conditions suitable for initiating the enzymatic reaction; and-   iii) measuring the fluorescence polarisation of the sample to    determine the extent of reaction.

Suitably, the enzyme may be selected from cleavage enzymes such asproteases that catalyse cleavage of the substrate into two or morefragments, thereby resulting in a decrease in fluorescence polarisation.Alternatively the enzyme may join two components, for example, a ligaseor a transferase, resulting in an increase in polarisation of thesample.

The fluorescent dyes according to the first embodiment of the firstaspect may be used in applications that include detecting anddistinguishing between various components in a mixture. In a fifthaspect, the present invention provides a set of two or more differentfluorescent dyes, each dye of said set of dyes having the formula (I):

wherein:

-   groups R³ and R⁴ are attached to atoms of the Z¹ ring structure and    groups R⁵ and R⁶ are attached to atoms of the Z² ring structure,    where Z¹ and Z² are hereinbefore defined;-   R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected from hydrogen,    halogen, amide, hydroxyl, cyano, amino, mono- or di-C₁-C₄    alkyl-substituted amino, sulphydryl, carbonyl, carboxyl, C₁-C₆    alkoxy, acrylate, vinyl, styryl, aryl, heteroaryl, C₁-C₂₀ alkyl,    aralkyl, sulphonate, sulphonic acid, quaternary ammonium, the group    -E-F and the group —(CH₂—)_(n)Y;-   R¹ and R² are independently selected from hydrogen, C₁-C₂₀ alkyl,    aralkyl, the group -E-F and the group —(CH₂—)_(n)Y;-   E is a spacer group having a chain from 1-60 atoms selected from the    group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus    atoms and F is a target bonding group;-   Y is selected from sulphonate, sulphate, phosphonate, phosphate,    quaternary ammonium and carboxyl; and n is an integer from 1 to 6;-   wherein each dye of said set has a distinguishably different    fluorescence lifetime compared with the lifetimes of the remaining    dyes of the set.

Preferably, in each dye of the set of dyes at least one of groups R¹,R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is the group -E-F where E and F arehereinbefore defined.

Preferably, the set of fluorescent dyes according to the invention willcomprise four different dyes, each dye of the set having a differentfluorescence lifetime. Preferably, each of the fluorescent dyes of theset of dyes exhibits a fluorescence lifetime in the range from 1 to 30nanoseconds, more preferably, in the range from 10 to 25 nanoseconds.

To distinguish between different fluorescent dyes in the set of dyes,the lifetime of the fluorescence emission of each of the dyes ispreferably separated by at least 0.5 nanoseconds.

The set of dyes may be used in a detection method wherein differentfluorescent dyes of the set of dyes are covalently bonded to a pluralityof different primary components, each primary component being specificfor a different secondary component, in order to identify each of aplurality of secondary components in a mixture of secondary components.The method comprises covalently binding different dyes of a set offluorescent dyes according to the fifth aspect of the invention todifferent primary components in a multicomponent mixture wherein eachdye of the set has a different fluorescence lifetime, compared with thefluorescence lifetimes of the remaining dyes of the set; adding thedye-labelled primary components to a preparation containing secondarycomponents under conditions to enable binding of at least a portion ofeach of said dye-labelled primary components to its respective secondarycomponent; and determining the presence or the amount of the boundsecondary component by measuring the fluorescence lifetime of each ofthe labelled primary component-secondary component complexes.

If required, any unreacted primary components may be removed orseparated from the preparation by, for example washing, to preventinterference with the analysis.

Preferably, a single wavelength of excitation can be used to excitefluorescence from two or more materials in a mixture, where eachfluoresces having a different characteristic fluorescent lifetime.

The set of fluorescent dyes according to the present invention may beused in any system in which the creation of a fluorescent primarycomponent is possible. For example, an appropriately reactivefluorescent dye according to the invention can be conjugated to a DNA orRNA fragment and the resultant conjugate then caused to bind to acomplementary target strand of DNA or RNA. Other examples of primarycomponent-secondary component complexes which may be detected includeantibodies/antigens and biotin/streptavidin.

The set of fluorescent dyes according to the present invention may alsobe advantageously used in fluorescent DNA sequencing based uponfluorescence lifetime discrimination of the DNA fragments. Briefly, eachone of a set of dyes, may be coupled to a primer. Various primers areavailable, such as primers from pUC/M13, λgt10, λgt11 and the like (seeSambrook et al, Molecular Cloning, A Laboratory Manual 2^(nd) Edition,Cold Spring Harbour Laboratory Press 1989). DNA sequences are clonedinto an appropriate vector having a primer sequence joined to the DNAfragment to be sequenced. After hybridisation to the DNA template,polymerase enzyme-directed synthesis of a complementary strand occurs.Different 2′,3′-dideoxynucleotide terminators are employed in each isdifferent sequencing reaction so as to obtain base-specific terminationof the chain extension reaction. The resulting set of DNA fragments areseparated by electrophoresis and the terminating nucleotide (and thusthe DNA sequence) is determined by detecting the fluorescence lifetimeof the labelled fragments. DNA sequencing may also be performed usingdideoxynucleotide terminators covalently labelled with the fluorescentdyes according to the present invention.

The non-fluorescent or substantially non-fluorescent dyes according tothe second embodiment of the first aspect may be used as the substratefor an enzyme and which upon reaction with the enzyme, yields afluorescent product.

Bacterial nitroreductases have been shown to catalyse the generalreaction set out below in Reaction Scheme 1.

where, in the presence of NADH or NADPH, one or more nitro groups on anorganic molecule may be reduced to a hydroxylamine (—NHOH) group whichmay subsequently be converted to an amine (—NH₂) group.

Thus, in a sixth aspect of the invention, there is provided a method ofincreasing the fluorescence of a non-fluorescent or substantiallynon-fluorescent dye of formula (I):

wherein:

-   groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² are hereinbefore    defined; and wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶,    R⁷ and R⁸ comprises at least one nitro group;-   characterised by the reduction of said at least one nitro group to    —NHOH or —NH₂

Suitably, reduction is by means of nitroreductase. This can be achievedby enzymatic conversion of a nitro group in a compound of formula (I) toa —NHOH or —NH₂ group by the action of the nitroreductase. Depending onthe structure of the dye, the intensity and/or lifetime of thefluorescence emission from the product of the nitroreductase reactionmay be increased so as to exhibit a lifetime typically in the range from1 to 30 nanoseconds. Moreover, the fluorescence lifetime characteristicsof the reaction product can be altered to suit the application by meansof additional substitutents, whilst retaining the nitro group(s) thatare involved in the reaction with nitroreductase. Thus, fluorescentreporters compatible for use with other fluors in multiplex systems canbe provided.

In a seventh aspect of the invention there is provided a method fordetecting nitroreductase enzyme activity in a composition comprising:

-   i) mixing said composition under conditions to promote    nitroreductase activity with a dye of formula (I):

wherein:

-   groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² are hereinbefore    defined and wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶,    R⁷ and R⁸ comprises at least one nitro group; and-   ii) measuring an increase in fluorescence wherein said increase is a    measure of the amount of nitroreductase activity.

Suitably, the measurement step (ii) may be a measure of fluorescenceintensity and/or fluorescence lifetime of the labelled product of thenitroreductase reaction.

In one embodiment of the seventh aspect, the composition comprises acell or cell extract. In principle, any type of cell can be used, i.e.prokaryotic or eukaryotic (including bacterial, mammalian and plantcells). Where appropriate, a cell extract can be prepared from a cell,using standard methods known to those skilled in the art (MolecularCloning, A Laboratory Manual 2^(nd) Edition (1989), Cold Spring HarbourLaboratory Press), prior to measuring fluorescence.

Typical conditions for nitroreductase activity comprise incubation ofthe composition in a suitable medium and the dye at approximately 37° C.in the presence of NADH and FMN.

In a eighth aspect of the invention there is provided an assay methodcomprising:

-   i) binding one component of a specific binding pair to a surface;-   ii) adding a second component of the specific binding pair under    conditions to promote binding between the components, said second    component being labelled with a nitroreductase enzyme;-   iii) adding a dye of formula (I):

wherein:

-   groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² are hereinbefore    defined and wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶,    R⁷ and R⁸ comprises at least one nitro group; and-   iv) detecting binding of the second component to the first component    by measuring an increase in fluorescence as a measure of bound    nitroreductase activity.

In a preferred embodiment of the eighth aspect, said specific bindingpair is selected from the group consisting of antibodies/antigens,lectins/glycoproteins, biotin/streptavidin, hormone/receptor,enzyme/substrate, DNA/DNA, DNA/RNA and DNA/binding protein.

Briefly, an in vitro assay method for the detection of antibody bindingmay be configured as follows. An antibody specific for an antigen ofinterest may be labelled by covalently linking it to an enzymaticallyactive nitroreductase. The labelled antibody can then be introduced intothe test sample containing the antigen under binding conditions. Afterwashing to remove any unbound antibody, the amount of bound antibody isdetected by incubating the sample with a substrate comprising a compoundof formula (I) having at least one nitro group under conditionspromoting nitroreductase activity and measuring an increase influorescence. The amount of fluorescence detected will be proportionalto the amount of nitroreductase-labelled antibody that has bound to theanalyte.

In an in vitro assay for detecting the binding of nucleic acids byhybridisation, either of the pair of target and probe nucleic acid isbound to a membrane or surface. The unbound partner is labelled withnitroreductase and incubated under hybridising conditions with the boundnucleic acid. Unbound, labelled nucleic acid is washed off and theamount of bound, labelled nucleic acid is measured by incubating themembrane or surface with a compound of formula (I) having at least onenitro group under conditions suitable for nitroreductase activity. Theamount of increase in fluorescence gives a measure of the amount ofbound labelled DNA.

Methods for coupling enzymes, such as nitroreductase, to otherbiomolecules, e.g. proteins and nucleic acids, are well known(Bioconjugate Techniques, Academic Press 1996). Coupling may be achievedby direct means, for example by use of a suitable bifunctionalcrosslinking agent (e.g. N-[γ-maleimidopropionic acid]hydrazine, Pierce)to covalently link the enzyme and binding partner. Alternatively,coupling may be achieved by indirect means, for example by separatelybiotinylating the enzyme and the binding partner using a chemicallyreactive biotin derivative, (e.g. N-hydroxysuccinimido-biotin, Pierce)and subsequently coupling the molecules through a streptavidin bridgingmolecule.

Cell based assays are increasingly attractive over in vitro biochemicalassays for use in high throughput screening (HTS). This is because cellbased assays require minimal manipulation and the readouts can beexamined in a biological context that more faithfully mimics the normalphysiological situation. Such in vivo assays require an ability tomeasure a cellular process and a means to measure its output. Forexample, a change in the pattern of transcription of a number of genescan be induced by cellular signals triggered, for example, by theinteraction of an agonist with its cell surface receptor or by internalcellular events such as DNA damage. The induced changes in transcriptioncan be identified by fusing a reporter gene to a promoter region whichis known to be responsive to the specific activation signal.

In fluorescence-based enzyme-substrate systems, an increase influorescence gives a measure of the activation of the expression of thereporter gene.

Accordingly, in a ninth aspect of the invention, there is provided anassay method which comprises:

-   i) contacting a host cell which has been transfected with a nucleic    acid molecule comprising expression control sequences operably    linked to a sequence encoding a nitroreductase with a dye of formula    (I):

wherein:

-   groups R¹, R², R³ ₁, R⁴, R⁵, R⁶ ₁, R⁷, R⁸, Z¹ and Z² are    hereinbefore defined and wherein at least one of groups R¹, R², R³,    R⁴, R⁵, R⁶, R⁷ and R⁸ comprises at least one nitro group; and-   ii) measuring an increase in fluorescence as a measure of    nitroreductase gene expression.

Suitably, the measurement step (ii) may be a measure of fluorescenceintensity and/or fluorescence lifetime of the labelled product of thenitroreductase reaction.

In one embodiment of the ninth aspect, the assay method is conducted inthe presence of a test agent whose effect on gene expression is to bedetermined.

Methods for using a variety of enzyme genes as reporter genes inmammalian cells are well known (for review see Naylor L. H., BiochemicalPharmacology, (1999), 58, 749-757). The reporter gene is chosen to allowthe product of the gene to be measurable in the presence of othercellular proteins and is introduced into the cell under the control of achosen regulatory sequence which is responsive to changes in geneexpression in the host cell. Typical regulatory sequences include thoseresponsive to hormones, second messengers and other cellular control andsignalling factors. For example, agonist binding to seven transmembranereceptors is known to modulate promoter elements including the cAMPresponsive element, NF-AT, SRE and AP1; MAP kinase activation leads tomodulation of SRE leading to Fos and Jun transcription; DNA damage leadsto activation of transcription of DNA repair enzymes and the tumoursuppressor gene p53. By selection of an appropriate regulatory sequencethe reporter gene can be used to assay the effect of added agents oncellular processes involving the chosen regulatory sequence under study.

For use as a reporter gene, the nitroreductase gene may be isolated bywell known methods, for example by amplification from a cDNA library byuse of the polymerase chain reaction (PCR) (Molecular Cloning, ALaboratory Manual 2^(nd) Edition, Cold Spring Harbour Laboratory Press(1989), pp 14.5-14.20). Once isolated, the nitroreductase gene may beinserted into a vector suitable for use with mammalian promoters(Molecular Cloning, A Laboratory Manual 2^(nd) Edition, Cold SpringHarbour Laboratory Press (1989), pp 16.56-16.57) in conjunction with andunder the control of the gene regulatory sequence under study. Thevector containing the nitroreductase reporter and associated regulatorysequences may then be introduced into the host cell by transfectionusing well known techniques, for example by use of DEAE-Dextran orCalcium Phosphate (Molecular Cloning, A Laboratory Manual 2^(nd)Edition, Cold Spring Harbour Laboratory Press (1989), pp 16.30-16.46).Other suitable techniques will be well known to those skilled in theart.

In another embodiment of the ninth aspect, the dye of formula (I)wherein groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, Z¹ and Z² arehereinbefore defined and wherein at least one of groups R¹, R², R³, R⁴,R⁵, R⁶, R⁷ and R⁸ comprises at least one nitro group, is permeable tocells. In this embodiment, preferably, at least one of groups R¹, R²,R³, R⁴, R⁵, R⁶, R⁷ and R⁸ comprises a cell membrane permeabilisinggroup. Membrane permeant compounds can be generated by maskinghydrophilic groups to provide more hydrophobic compounds. The maskinggroups can be designed to be cleaved from the substrate within the cellto generate the derived fluorogenic substrate intracellularly. Becausethe substrate is more hydrophilic than the membrane permeant derivative,it is then trapped in the cell. Suitable cell membrane permeabilisinggroups may be selected from acetoxymethyl ester, which is readilycleaved by endogenous mammalian intracellular esterases (Jansen, A. B.A. and Russell, T. J., J.Chem.Soc., (1965), 2127-2132 and Daehne W. etal. J.Med.Chem., (1970) 13, 697-612) and pivaloyl ester (Madhu et al.,J. Ocul.Pharmacol.Ther., (1998), 14(5), 389-399) although other suitablegroups will be recognised by those skilled in the art.

Typically, to assay the activity of a test agent to activate cellularresponses via the regulatory sequence under study, cells transfectedwith the nitroreductase reporter are incubated with the test agent,followed by addition of a dye of formula (I) wherein at least one ofgroups R¹, R², R³, R⁴ and R⁵ in said dye comprises at least one nitrogroup, said compound being made cell permeant. After an appropriateperiod required for conversion of the substrate to a form exhibitingfluorescence characteristics, the fluorescence intensity and/orfluorescence lifetime from the cells is measured at an emissionwavelength appropriate for the chosen dye. Measurement of fluorescencemay be readily achieved by use of a range of detection instrumentsincluding fluorescence microscopes (e.g. LSM 410, Zeiss), microplatereaders (e.g. CytoFluor 4000, Perkin Elmer), CCD imaging systems (e.g.LEADseeker™, Amersham Pharmacia Biotech) and Flow Cytometers (e.g.FACScalibur, Becton Dickinson).

The measured fluorescence is compared with fluorescence from controlcells not exposed to the test agent and the effects, if any, of the testagent on gene expression modulated through the regulatory sequence, isdetermined from the ratio of fluorescence in the test cells to thefluorescence in the control cells. Where appropriate, a cell extract canbe prepared using conventional methods.

Suitable means for expressing a nitroreductase enzyme include anexpression plasmid or other expression construct. Methods for preparingsuch expression constructs are well known to those skilled in the art.

In a tenth aspect of the present invention, there is provided a dye offormula (I):

wherein:

-   groups R³ and R⁴ are attached to atoms of the Z¹ ring structure and    groups R⁵ and R⁶ are attached to atoms of the Z² ring structure;-   Z¹ and Z² independently represent the atoms necessary to complete    one ring, two fused ring, or three fused ring aromatic or    heteroaromatic systems, each ring having five or six atoms selected    from carbon atoms and optionally no more than two atoms selected    from oxygen, nitrogen and sulphur;-   at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is the    group -E-F where E is a spacer group having a chain from 1-60 atoms    selected from the group consisting of carbon, nitrogen, oxygen,    sulphur and phosphorus atoms and F is a target bonding group; and,-   when any of said groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is not said group    -E-F, said remaining groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are    independently selected from hydrogen, halogen, amide, hydroxyl,    cyano, nitro, amino, mono- or di-C₁-C₄ alkyl-substituted amino,    sulphydryl, carbonyl, carboxyl, C₁-C₆ alkoxy, acrylate, vinyl,    styryl, aryl, heteroaryl, C₁-C₂₀ alkyl, aralkyl, sulphonate,    sulphonic acid, quaternary ammonium and the group —(CH₂—)_(n)Y; and,-   when any of groups R¹ and R² is not said group -E-F, said remaining    groups R¹ and R² are independently selected from hydrogen, mono- or    di-nitro-substituted benzyl, C₁-C₂₀ alkyl, aralkyl and the group    —(CH₂—)_(n)Y;-   E is a spacer group having a chain from 1-60 atoms selected from the    group consisting of carbon, nitrogen, oxygen, sulphur and phosphorus    atoms and F is a target bonding group;-   Y is selected from sulphonate, sulphate, phosphonate, phosphate,    quaternary ammonium and carboxyl; and n is an integer from 1 to 6;-   provided that at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and    R⁸ is a water solubilising group.

Preferably, the target bonding group F comprises a reactive group forreacting with a functional group on a target material, or a functionalgroup for reacting with a reactive group on a target material. Preferredreactive groups may be selected from carboxyl, succinimidyl ester,sulpho-succinimidyl ester, isothiocyanate, maleimide, haloacetamide,acid halide, hydrazide, vinylsulphone, dichlorotriazine andphosphoramidite. Preferred functional groups may be selected fromhydroxy, amino, sulphydryl, imidazole, carbonyl including aldehyde andketone, phosphate and thiophosphate.

Preferably, the spacer group E is selected from:

-   —(CHR′)_(p)—-   —{(CHR′)_(q)—O—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)—-   —{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)—    where R′ is hydrogen, C₁-C₄ alkyl or aryl, which may be optionally    substituted with sulphonate, Ar is phenylene, optionally substituted    with sulphonate, p is 1-20, preferably 1-10, q is 0-10, r is 1-10    and s is 1-5.

Dyes according to the tenth aspect may contain a polymerizable groupsuitable for the formation of a polymer containing the dye. Suitablepolymerizable groups are selected from acrylate, methacrylate andacrylamide. Polymerization may be carried out with a suitablyderivatized compound of this invention used in conjunction with a secondpolymerizable monomer starting material, such as styrene orvinyltoluene, to form a copolymer containing the dye. The dyes of thepresent invention need not have a polymerisable group, for example, thedye may be incorporated during polymerisation or particle formation ormay be absorbed into or onto polymer particles.

The dyes of formula (I) may be prepared by a process comprising reactingdiethyl succinyl-succinate with an appropriately substituted anilineaccording to published methods (see Jaffe, E. E. and Marshall, W. J.,U.S. Pat. No. 3,386,843; Jaffe, E. E. and Ehrich, F. F. U.S. Pat. No.3,873,548). For example, heating diethyl succinyl-succinate with4-aminobenzoic acid affordsdiethyl-2,5-di(4-carboxyanilino)-3,6-dihydroterphthalate. Furtherheating in a high boiling solvent affords2,9-dicarboxy-6,13-dihydroquinacridone. Oxidation with sodium3-nitrobenzene sulphonate gives 2,9-dicarboxyquinacridone. Alternativemethods of synthesising quinacridone and its derivatives are disclosedby Bender, H. et al (U.S. Pat. No. 4,956,464), whereby2,5-dianilino-3,6-dihydroterephthalic acid derivatives may be cyclizedand dehydrogenated at 500-600° C. Maki, H. et al (U.S. Pat. No.5,659,036) describe methods for preparing quinacridone derivatives inwhich alkyl esters of 1,4-cyclohexadione-2,5-dicarboxylic acid may bereacted with an appropriately substituted aromatic amine and theresultant 2,5-di(arylamino)-3,6-dihydroterephthalic acid dialkyl esterderivative may be cyclized to a 6,13-dihydroquinacridone which is thenoxidized with a nitrobenzene sulphonic acid to give the quinacridone.

It will be readily appreciated that certain dyes of formula (I) may beuseful as intermediates for conversion to other dyes of formula (I) bymethods well known to those skilled in the art. Likewise, certain of theintermediates may be useful for the synthesis of dyes of formula (I).The compounds of the present invention may be synthesized by the methodsdisclosed herein. Derivatives of the dyes having a particular utilityare prepared either by selecting appropriate precursors or by modifyingthe resultant compounds by known methods to include functional groups ata variety of positions. As examples, the dyes of the present inventionmay be modified to include certain reactive groups for preparing afluorescent labelling reagent, or charged or polar groups may be addedto enhance the solubility of the compound in polar or non-polar solventsor materials. As examples of conversions an ester may be converted to acarboxylic acid or may be converted to an amido derivative. Groups R¹ toR⁸ may be chosen so that the compounds of the present invention havedifferent fluorescence characteristics, thereby providing a number ofrelated dyes which can be used in multiparameter analyses wherein thepresence and quantity of different compounds in a single sample may bedifferentiated based on the wavelengths and lifetimes of a number ofdetected fluorescence emissions. The dyes of the present invention maybe made soluble in aqueous, other polar, or non-polar media containingthe material to be labelled by appropriate selection of R-groups.

The invention is further illustrated by reference to the followingexamples and figures in which:

FIG. 1 is a plot showing the absorbance and emission spectrum of6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid;

FIG. 2 shows the fluorescence lifetime decay plots of three dyesaccording to the present invention;

FIG. 3 is a lifetime decay plot of6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl)hexanoicacid and of its conjugate with ovalbumin;

FIG. 4 is a plot showing trypsin cleavage of a conjugate of albumin with6-{12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid monitored by fluorescence polarisation.

Cy™ and LEADseeker™ are trademarks of Amersham Pharmacia Biotech UKLimited.

EXAMPLES 1. 2-Bromo-5,12-dihexylquinacridone (A) and2,9-dibromo-5,12-dihexylquinacridone (B)

1.1 5,12-Dihexylquinacridone

A 25 ml RB flask was charged with quinacridone (Dojindo;156 mg, 0.5mmol) and anhydrous N,N-dimethylformamide (5 ml). The flask was purgedwith nitrogen and set stirring. To the mixture was added sodium hydride(60 wt % dispersion in oil, 48 mg, 1.2 mmol) and the mixture left tostir. A blue colour slowly developed in the liquid phase but much of thesolids remained undissolved after 2 hrs; dissolution was achieved by theaddition of dimethyl sulphoxide (3 ml) to give a deep blue solutionafter a further 1 hr. At this point 1-iodohexane (295 μl, 2.0 mmol) wasadded and the mixture left to stir for 3 days. During this time the bluecolour was completely discharged and an orange solid precipitated.

The mixture was quenched into 0.5M aqueous hydrochloric acid, the solidcollected by filtration and washed with excess water. The damp solid wasthen dried by dissolution into dichloromethane containing anhydrousmagnesium sulfate; the slurry was filtered and the brown-orange filtrateevaporated under vacuum to give the crude product. This was purified byflash chromatography (silica. 0-2.5% ethyl acetate in dichloromethane);pure fractions were pooled, filtered and evaporated, then trituratedwith diethyl ether to give an orange powder. Yield=161 mg (67%). λ_(max)(CH₂Cl₂)=526, 493 nm. δ_(H) (200 MHz, CDCl₃) 0.95 (6H, t), 1.20-1.65(12H, m), 2.01 (4H, m), 4.52 (4H, app t), 7.28 (2H, app t), 7.52 (2H,app d), 7.76 (2H, td), 8.58 (2H, dd) and 8.79 (2H, s).

Mass spectrum: (ES+) 481 (M+H), 503 (M+Na). Accurate mass:(M+H)=C₃₂H₃₇N₂O₂, requires 481.2855. Found 481.2844 (−2.3 ppm).

1.2 2-Bromo-5,12-dihexylquinacridone and2,9-dibromo-5,12-dihexylquinacridone

5,12-Dihexylquinacridone (60 mg, 125 μmol) was mixed with ethanol (2.5ml) and benzyltrimethylammonium tribromide (97 mg, 250 μmol). Theresulting slurry was stirred at ambient temperature for 24 hrs, thenheated under reflux for 16 hrs to give limited reaction. Addition ofchloroform (2.5 ml) dissolved all solids to give an orange solution,continued reflux gave moderate generation of mono- and di-brominatedproducts. After evaporation of solvent the products were isolated byflash chromatography (silica, dichloromethane) to give pure samples.

2-Bromo-5,12-dihexylquinacridone (A): λ_(max) (CH₂Cl₂)=526, 493 nm.δ_(H) (200 MHz, CDCl₃) 0.95 (6H, m), 1.3-1.7 (12H, m), 1.9-2.1 (4H, m),4.4-4.5 (4H, m), 7.25 (1H, m), 7.36 (1H, d), 7.48 (1H, d), 7.70-7.80(2H, m), 8.52 (1H, dd), 8.61 (1H, s), 8.67 (1H, s) and 8.71 (1H, s).Mass spectrum (DEI+): 558+560 (ratio 1:1) (M+).

2,9-Dibromo-5,12-dihexylquinacridone (B): λ_(max) (CH₂Cl₂)=532, 498 nm.δ_(H) (200 MHz, CDCl₃) 0.95 (6H, t), 1.3-1.7 (12H, m), 1.8-2.0 (4H, m),4.5 (4H, t), 7.36 (2H, d), 7.78 (2H, dd), 8.58 (2H, d) and 8.64 (2H, s).Mass spectrum (DEI+): 636+638+640 (ratio 1:2:1) (M+).

2. Quinacridone-2,9-disulphonic acid, (di-potassium salt)

Quinacridone (200 mg, 0.645 mM) was placed in a round bottomed flaskfitted with a magnetic stirrer and air condenser. The solid wasdissolved in 98% sulphuric acid (2 ml) and heated to 110° C. for 6 hrunder a nitrogen atmosphere. TLC (RP C-18 50/50 methanol/water) showedthat all the starting material had been converted to a single fastmoving component (visualised under long wavelength uv light). Thereaction was dripped onto 10 ml ice to give a dark red solution. Thiswas neutralised with solid potassium hydrogen carbonate to give a darkred precipitate. This was collected by centrifugation and thesupernatant discarded.

Recrystallisation from water gave 0.21 g (0.386 Mm, 60%) of red solididentified as the di-potassium salt of quinacridone-2,9-disulphonicacid. Mass Spec(ES+). MH⁺ 473.1; MK₂ ⁺ 550.5. MPt >300° C.

λ_(max)(ab) 499, 527 nm (water); λ_(max)(em) 560,590 nm (water).

3. Quinacridone-2,4,9,11-tetrasulphonic acid (tetra-potassium salt)

Quinacridone (500 mg, 1.61 mM) was placed in a round bottomed flaskfitted with a magnetic stirrer and air condenser. The solid wasdissolved in 20% oleum (5 mL) and heated to 110° C. for 20 hr under anitrogen atmosphere. TLC (RP C-18 50/50 methanol/water) showed that allthe starting material had been converted to a major fast movingcomponent with two minor faster moving and one minor slower movingcomponent (visualised under long wavelength uv light). The reaction wasdripped onto 10 ml ice to give a dark red solution. This was neutralisedwith solid potassium hydrogen carbonate to give an orange precipitate.This was collected by centrifugation and the supernatant discarded.

Recrystallisation twice from water gave 1.1 gm of an orange solididentified as the tetra-potassium salt ofquinacridone-2,4,9,11-tetrasulphonic acid. Mass Spec (ES+). MH⁺ 632.9.

NMR (200 MHz, D₂O): δ 8.94 (doublet), δ 8.81 (single) δ 8.63 (doublet)1:1:1. MPt >300° C. λ_(max)(ab) 499,527 nm (water); λ_(max)(em) 552 nm(water).

4.O—(N-Succinimidyl)-6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate

4.16-{7,14-Dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid ethyl ester

Quinacridone-2,9-disulphonic acid (200 mg) was dried by repeated rotaryevaporation with dry DMF. The material was then dissolved in 10 ml dryDMSO to give an orange solution. Potassium t-butoxide (45 mg, 0.4 mM)was added, the solution immediately turned blue. The solution was thenstirred magnetically for 30 mins and then ethyl 6-bromohexanoate (70 μl,0.4 mM) was added and the solution stirred under a nitrogen atmospherefor a further 24 hours. TLC (RP C-18 50/50 methanol/water) showed thatapproximately half of the starting material had been converted to aslightly slower running component. Further reaction time did notincrease the amount of product. Mass spec. showed a peak at 692corresponding to MK₂ ⁺ for the required product. A further peak at 550corresponds to MK₂ ⁺ for the starting material. The solvent was removedby rotary evaporation to leave a red solid. No further attempt was madeto purify this material which was used as such in subsequent reactions.

4.26-{7,14-Dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid

The material from the above reaction was dissolved in 8 ml of a mixtureof 1M hydrochloric acid: glacial acetic acid (1:3) and heated at 100° C.for two hours in a flask fitted with a reflux condenser and under anitrogen atmosphere. Mass spec. showed the disappearance of the peak at692 as described in the previous example and the appearance of a peak at664 corresponding to MK₂ ⁺ for6-(7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl)hexanoicacid. A further peak at 550 corresponds to MK₂ ⁺ forquinacridone-2,9-disulphonic acid. TLC (as described above showed asingle spot). The solvents were removed by rotary evaporation to give ared solid. No attempt was made to purify this material which was thenused in the next reaction.

4.3O—(N-Succinimidyl)-6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate

The material from the previous reaction was dried by repeated rotaryevaporation from DMF. The solid was then dissolved in 10 ml dry DMSO and200 μl diisopropylethylamine was added followed byO-(succinimidyl)-N,N,N′,N′-tetramethylene uronium hexafluorophosphate(HSPyU, 85 mg). The mixture was stirred at ambient temperature under anitrogen atmosphere for 2 hrs. TLC (as described above) showed thepresence of two spots. Mass spec. showed the disappearance of the peakat 692 as described in the previous example and the appearance of a peakat 684 corresponding to MH⁺ forO—(N-succinimidyl)-6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate.A further peak at 550 corresponds to MK₂ ⁺ forquinacridone-2,9-disulphonic acid. The solvent was removed by rotaryevaporation to leave a red gum. Trituration with ethyl acetate gave 74mg of a red solid.

5.O—{N-succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate

5.16-{12-ethyl-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, ethyl ester

Quinacridone (1.56 gm; 5.0 mmol) was suspended in anhydrousdimethylformamide (15 ml) and anhydrous dimethyl sulphoxide (15 ml)under a nitrogen atmosphere. Sodium hydride (60% suspension in oil; 240mg; 6.0 mmol) was added and the mixture stirred until effervescencestopped. More sodium hydride (240 mg; 6.0 mmol) was added and themixture stirred for 10 minutes when effervescence had ceased. Thereaction was heated to 60° C. for 1 hour. Ethyl 6-bromohexanoate (890μl; 5.0 mmol) was added to the dark green solution and the mixturestirred overnight at 60° C. Iodoethane (1.0 ml; 12.5 mmol) was thenadded and the mixture stirred for 2 hours at 60° C. The dark orange-redsolution was allowed to cool, then the mixture was poured into water(300 ml). The solid was filtered off, washed with water and air dried.The solid was then dissolved in dichloromethane (300 ml) and anhydrousmagnesium sulphate added. The mixture was filtered and the solventremoved by rotary evaporation to give a red solid. This was purified byflash chromatography (silica, 15% ethyl acetate/dichloromethane) to give1.04 gm (43%) of the diethyl ester of6-{12-ethyl-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid.

5.26-{12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid

The diethyl ester of6-{12-ethyl-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid (241 mg; 0.5 mmol) was dissolved in conc. sulphuric acid (5 ml) andthe purple solution heated at 110° C. overnight under an atmosphere ofnitrogen. The reaction was allowed to cool and then poured onto ice (˜20gm). The solution was neutralised with 40% w/v sodium hydroxide solutionto give a bright red solution. This was acidified with glacial aceticacid when a orange-red precipitate formed. This was collected bycentrifugation, then dissolved in 0.1% trifluoroacetic acid (TFA) inwater. The solution was purified by reverse phase HPLC. Vydac C18semi-preparative column, water to acetonitrile gradient (both containing0.1% v/v TFA), flow 5 ml/minute, detection at 530 nm. Purified materialwas pooled, evaporated to dryness under vacuum and then dried undervacuum over phosphorous pentoxide to give 300 mg (97%) of6-{12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid as a dark red solid.

δ_(H) (200 MHz, CD₃OD +D₂O) 1.48(3H, t), 1.8(6H, m), 2.38(2H, m),4.52(4H, m), 7.59(2H, m), 8.10(2H, d), 8.60(2H, m), 8.89(2H, d).Accurate mass: (M+H)=C₂₈H₂₇N₂O₁₀S₂, requires 615.1107. Found 615.1089(2.9 ppm). λ_(max)(ab) 294 nm (ε=65,800/M⁻¹cm⁻¹); 506 nm(εs=5590/M⁻¹cm⁻¹); 536 nm (ε=4790/M⁻¹cm⁻¹). (PBS buffer) λ_(max)(em) 563nm (PBS buffer)

5.3O—{N-Succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate.

6-{12-Ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid (15 mg; 241 μmol), O—(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (8 mg; 27 μmol), anhydrous dimethyl sulphoxide (500μl) and diisopropylethylamine (17.5 μl) were mixed to give an orangesolution. This was left for 30 minutes when TLC (RP₁₈ 30:70water:methanol) showed that the starting material had been converted toa slower running component identified asO—(N-succinimidyl-6-{12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoateby mass spectroscopy.

Mass spectrum: (ES+) (M+H) 712

6.6-{2,9-Dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid

6.1 Dimethyl2,5-bis{(4-bromophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate

Dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (4.56 gm; 20 mmol) andmethanol (100 ml) were heated to boiling, then 4-bromoaniline (6.88 gm;40 mmol) was added followed by conc. hydrochloric acid (200 μl). Themixture was refluxed for 5 hours under a nitrogen atmosphere. On coolinga cream solid precipitated out which was collected by filtration, washedwith methanol and dried under vacuum to give 10.18 gm (95%) of dimethyl2,5-bis{(4-bromophenyl)amino}cyclohexa-1,4-diene-1,4-dicaboxylate.

6.2 2,5-Bis{(4-bromophenyl)amino}terephthalic acid

Dimethyl2,5-bis{(4-bromophenyl}amino]cyclohexa-1,4-diene-1,4-dicarboxylate (5.36gm; 10 mmol) the sodium salt of 3-nitrobenzenesulphonic acid (2.3 gm; 10mmol), ethanol (50 ml) and 1.0M sodium hydroxide (30 ml) were heated toreflux for 7 hours under a nitrogen atmosphere. The bright yellowsolution was allowed to cool and water (120 ml) was added. The mixturewas acidified with conc. hydrochloric acid when a magenta solidprecipitated out. This material was filtered off, washed with water anddried under vacuum over phosphorous pentoxide to give 4.84 gm (96%) of2,5-bis{(4-bromophenyl)amino}terephthalic acid.

6.3 2,9-Dibromoquinacridone

2,5-Bis{(4-bromophenyl)amino}terephthalic acid (4.0 gm; 7.9 mmol) andpolyphosphoric acid (34 gm) were heated at 150° C. for 4 hours under anitrogen atmosphere. The mixture was allowed to cool and then pouredinto iced water (100 ml) when a magenta solid was formed. The solid wasfiltered off, washed with water, then methanol and dried under vacuumover phosphorous pentoxide to give 3.68 gm (99%)2,9-dibromoquinacridone.

6.46-{2,9-dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

2,9-Dibromoquinacridone (2.35 gm; 5.0 mmol) was suspended in anhydrousdimethylformamide (15 ml) under a nitrogen atmosphere. Sodium hydride(60% suspension in oil; 480 mg; 12 mmol) was added and the mixturestirred until effervescence stopped. Anhydrous dimethyl sulphoxide (25ml) was added. The reaction was heated to 70° C. for 2 hour. Ethyl6-bromohexanoate (2.67 ml; 15 mmol) was added to the dark green solutionand the mixture stirred overnight at 50° C. The dark blue solution wasallowed to cool, then the mixture was poured into water (200 ml) andacidified with conc. hydrochloric acid. The solid was filtered off,washed with water and air dried. This was purified by flashchromatography (silica. 5-20% ethyl acetateldichloromethane) to give1.74 gm (46%) of6-{2,9-dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester.

δ_(H) (200 MHz, CDCl₃) 1.25(6H, t), 1.80(12H, m), 2.39(4H, t), 4.15(4H,dd), 4.39(4H, t), 7.24(2H, d), 7.70(2H, dd), 8.42(4H, s). λ_(max)(ab)493 nm, 527 nm λ_(max)(em) 560 nm, 600 nm. (Dichloromethane).

6.56-{2,9-dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid

6-{2,9-Dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester (1.0 gm) was dissolved in glacial acetic acid (20ml) to give a deep magenta solution. 1.0M hydrochloric acid (10 ml) wasadded and the mixture heated to reflux for 5 hours. The reaction wasallowed to cool, the red precipitate filtered off, washed with aceticacid and then diethyl ether and dried under vacuum over phosphorouspentoxide to give 0.86 gm (93%) of6-{2,9-dibromo-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid.

Mass spectrum: (ES+) (M+H) 753, 755, 757. λ_(max)(ab) 499 nm, 533 nm.λ_(max)(em) 552 nm, 595 nm. (methanol)

7.6-{2,9-Dichloro-12-(5-carboxypentyI)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

7.1 Dimethyl2,5-bis{(4-chlorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate

Dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (4.5 6 gm; 20 mmol) andmethanol (100 ml) were heated to boiling; then 4-chloroaniline (5.36 gm;42 mmol) was added followed by conc. hydrochloric acid (200 μl). Themixture was refluxed for 5 hours under a nitrogen atmosphere. Oncooling, a cream solid precipitated out which was collected byfiltration, washed with methanol and dried under vacuum to give 8.62 gm(96%) of dimethyl2,5-bis{(4-chlorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate.

7.2 2,5-Bis{(4-chlorophenyl)amino}terephthalic acid

Dimethyl2,5-bis{(4-chlorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate(4.47 gm, 10 mmol), the sodium salt of 3-nitrobenzenesulphonic acid (2.3gm; 10 mmol), ethanol (70 ml) and 1.0M sodium hydroxide (40 ml) wereheated to reflux overnight under a nitrogen atmosphere. The brightyellow solution was allowed to cool and water (120 ml) was added. Themixture was acidified with conc. hydrochloric acid when a red solidprecipitated out. This material was filtered off, washed with water anddried under vacuum over phosphorous pentoxide to give 4.0 gm (96%) of2,5-bis{(4-chlorophenyl)amino}terephthalic acid.

λ_(max)(ab) 308 nm, 379 nm. (0.1M sodium hydroxide) Mass spectrum (ES+)(M+H) 417.

7.3 2,9-Dichloroquinacridone

2,5-Bis{(4-chlorophenyl)amino}terephthalic acid (3.35 gm; 8 mmol) andpolyphosphoric acid (30 gm) were heated at 150° C. for 3 hours under anitrogen atmosphere. The mixture was allowed to cool and then pouredinto iced water (200 ml) when a magenta solid precipitated out. This wasfiltered off, washed with water and methanol, then dried under vacuumover phosphorous pentoxide to give 3.1 gm (100%) of2,9-dichloroquinacridone.

Mass spectrum (ES+) (M+H) 381

7.46-{2,9-Dichloro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

2,9-Dichloroquinacridone (381 mg; 1.0 mmol) was suspended in anhydrousdimethylformamide (4 ml) under a nitrogen atmosphere. Sodium hydride(60% suspension in oil; 100 mg; 2.40 mmol) was added and the mixturestirred until effervescence stopped. Anhydrous dimethyl sulphoxide (7ml) was added. The reaction was heated to 70° C. for 1 hour. Ethyl6-bromohexanoate (535 μl; 3.0 mmol) was added to the dark green solutionand the mixture stirred overnight at 70° C. The dark orange-red solutionwas allowed to cool, then the mixture was poured into water (150 ml) and1.0M hydrochloric acid (10 ml). The solid was filtered off, washed withwater and air dried. This was purified by flash chromatography (silica.20% ethyl acetate/dichloromethane) to give a red oil which crystallisedon triturating with diethyl ether to give 205 mg (31%) of6-{2,9-dichloro-12-(5-carboxy-pentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester.

Mass spectrum (ES+) (M+H) 665 δ_(H) (200 MHz, CDCl₃) 1.27(6H, t),1.80(12H, m), 2.39(4H, t), 4.15(4H, dd), 4.46(4H, t), 7.40(2H, d).7.64(2H, dd), 8.40(2H, d), 8.6(2H, s) λ_(max)(ab) 464 nm, 493 nm, 528nm. λ_(max)(em) 560 nm, 600 nm. (Dichloromethane).

8.6-{2,9-Difluoro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

8.1 Dimethyl2,5-bis{(4-fluorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate

Dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (9.12 gm; 40 mmol) andmethanol (200 ml) were heated to boiling, then 4-fluoroaniline (8.35 ml(9.78 gm); 42 mmol) was added followed by conc. hydrochloric acid (400μl). The mixture was refluxed for 3 hours under a nitrogen atmosphere.On cooling a yellow solid precipitated out which was collected byfiltration, washed with methanol and dried under vacuum to give 15.8 gm(96%) of dimethyl2,5-bis{(4-fluorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate.

8.2 2,5-Bis{(4-fluorophenyl)amino}terephthalic acid

Dimethyl2,5-bis{(4-fluorophenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate(6.21 gm, 15 mmol), the sodium salt of 3-nitrobenzenesulphonic acid (3.6gm; 16 mmol), ethanol (90 ml) and 1.0M sodium hydroxide (50 μl) wereheated to reflux overnight under a nitrogen atmosphere. The brightyellow solution was allowed to cool and water (120 ml) was added. Themixture was acidified with conc. hydrochloric acid when a red solidprecipitated out. This material was filtered off, washed with water anddried under vacuum over phosphorous pentoxide to give 5.6 gm (97%) of2,5-bis{(4-fluorophenyl)amino}terephthalic acid λ_(max)(ab) 295 nm, 380nm. (0.1M sodium hydroxide)

8.3 2,9-Difluoroquinacridone

2,5-Bis{(4-fluorophenyl)amino}terephthalic acid (5.0 gm; 13 mmol) andpolyphosphoric acid (˜50 gm) were heated at 150° C. for 3 hours under anitrogen atmosphere. The mixture was allowed to cool and then pouredinto iced water (200 ml) when a magenta solid precipitated out. This wasfiltered off, washed with water and then methanol, then dried undervacuum over phosphorous pentoxide to give 4.5 gm (99%) of2,9-difluoroquinacridone.

Mass spectrum (ES+) (M+H) 349

8.46-{2,9-Difluoro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

2,9-Difluoroquinacridone (350 mg; 1.0 mmol) was suspended in anhydrousdimethylformamide,(4 ml) under a nitrogen atmosphere. Sodium hydride(60% suspension in oil; 100 mg; 2.40 mmol) was added and the mixturestirred until effervescence stopped. The reaction was heated to 70° C.for 1 hour. Ethyl 6-bromohexanoate (535 μl; 3.0 mmol) was added to thedark green solution and the mixture stirred overnight at 70° C. The darkorange-red solution was allowed to cool; then the mixture was pouredinto water (150 ml) and 1.0M hydrochloric acid. The solid was filteredoff, washed with water and air dried. This was purified by flashchromatography (silica. 20% ethyl acetate/dichloromethane) to give a redoil which crystallised on triturating with diethyl ether to give 171 mg(27%) of6-{2,9-difluoro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester.

δ_(H) (200 MHz, CDCl₃) 1.27(6H, t), 1.80(12H, m), 2.39(4H, t), 4.15(4H,dd), 4.48(4H, t), 7.46(4H, dd), 8.12(2H, d), 8.61(2H, s). λ_(max)(ab)495 nm, 533 nm. λ_(max)(em) 570 nm, 605 nm. (Dichloromethane) Massspectrum (ES+) (M+H) 633.

9.6-{2,9-dimethyl-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

9.1Dimethyl2,5-bis{(4-methylphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate

Dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (4.56 gm; 20 mmol) andmethanol (100 ml) were heated to boiling; then 4-methylaniline (4.5 gm;42 mmol) was added followed by conc. hydrochloric acid (200 μl). Themixture was refluxed for 5 hours under a nitrogen atmosphere. On coolinga cream solid precipitated out which was collected by filtration, washedwith methanol and dried under vacuum to give 7.92 gm (97%) of dimethyl2,5-bis{(4-methylphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate.

9.2 2,5-Bis{(4-methylphenyl)amino}terephthalic acid

Dimethyl2,5-bis{(4-methylphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate (5.1gm, 12.5 mmol), sodium salt of 3-nitrobenzenesulphonic acid (2.93 gm; 13mmol), ethanol (75 ml) and 1.0M sodium hydroxide (40 ml) were heated toreflux overnight under a nitrogen atmosphere. The bright yellow solutionwas allowed to cool and water (120 ml) was added. The mixture wasacidified with conc. hydrochloric acid when a purple solid precipitatedout. This material was filtered off, washed with water and dried undervacuum over phosphorous pentoxide to give 4.53 gm is (96%) of2,5-bis{(4-chlorophenyl)amino}terephthalic acid.

λ_(max)(ab) 299 nm, 386 nm. (0.1M sodium hydroxide).

9.3 2,9-Dimethylquinacridone

2,5-Bis{(4-methylphenyl)amino}terephthalic acid (3.76 gm; 10 mmol) andpolyphosphoric acid (40 gm) were heated at 150° C. for 2.5 hours under anitrogen atmosphere. The mixture was allowed to cool and then pouredinto iced water (100 ml) when a magenta solid precipitated out. This wasfiltered off, washed with water and then methanol, then dried undervacuum over phosphorous pentoxide to give 3.12 gm (92%) of2,9-dimethylquinacridone.

9.46-{2,9-Dimethyl-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

2,9-Dimethylquinacridone (680 mg; 2.0 mmol) was suspended in a mixtureof anhydrous dimethylformamide (10 ml) and anhydrous dimethyl sulphoxide(10 ml) under a nitrogen atmosphere. Sodium hydride (60% suspension inoil; 200 mg; 5.0 mmol) was added and the mixture stirred untileffervescence stopped. The reaction was heated to 70° C. for 1 hour.Ethyl 6-bromohexanoate (1.07 ml (1.34 gm); 6.0 mmol) was added to thedark blue-green solution and the mixture stirred overnight at 60° C. Thedark orange-red solution was allowed to cool, then the mixture waspoured into water (150 ml) and 1.0M hydrochloric acid (30 ml). The solidwas filtered off, washed with water and air dried. This was purified byflash chromatography (silica, 5-25% ethyl acetate/dichloromethane) togive a red oil which crystallised on triturating with diethyl ether togive 780 mg (62%) of6-{2,9-dimethyl-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester.

δ_(H) (200 MHz, CDCl₃) 1.24 (6H, t), 1.80(12H, m), 2.38(10H, m),4.15(4H, dd), 4.45(4H, t), 7.30(2H dd), 7.47(2H, dd), 8.21(2H, d),8.57(2H, s). λ_(max)(ab) 495 nm, 530 nm λ_(max)(em) 545 nm.(Dichloromethane).

10.6-{2,9-Dimethoxy-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

10.1 Dimethyl2,5-bis{(4-methoxyphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate

Dimethyl 1,4-cyclohexanedione-2,5-dicarboxylate (9.12 gm; 40 mmol) andmethanol (200 ml) were heated to boiling, then 4-methoxyaniline (10.84gm; 88 mmol) was added followed by conc. hydrochloric acid (400 μl). Themixture was refluxed for 2.5 hours under a nitrogen atmosphere. Oncooling, an orange solid precipitated out which was collected byfiltration, washed with methanol and dried under vacuum to give 17.0 gm(97%) of dimethyl2,5-bis{(4-methoxyphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate.

10.2 2,5-Bis{(4-methoxyphenyl)amino}terephthalic acid

Dimethyl2,5-bis{(4-methoxyphenyl)amino}cyclohexa-1,4-diene-1,4-dicarboxylate(6.58 gm, 15 mmol), the sodium salt of 3-nitrobenzenesulphonic acid (3.6gm; 16 mmol), ethanol (90 ml) and 1.0M sodium hydroxide (50 ml) wereheated to reflux overnight under a nitrogen atmosphere. The orangesolution was allowed to cool and water (120 ml) was added. The mixturewas acidified with conc. hydrochloric acid when a purple solidprecipitated out. This material was filtered off, washed with water,then 25% ethanol/water and dried under vacuum over phosphorous pentoxideto give 6.0 gm (98%) of 2,5-bis{(4-methoxyphenyl)amino}terephthalicacid.

λ_(max)(ab) 299 nm, 392 nm. (0.1M sodium hydroxide).

10.3 2,9-Dimethoxyquinacridone

2,5-Bis{(4-methoxyphenyl)amino}terephthalic acid (1.02 gm; 2.5 mmol) andpolyphosphoric acid (10 gm) were heated at 160° C. for 15 minutes undera nitrogen atmosphere. The mixture was allowed to cool and then pouredinto iced water (200 ml) when a purple solid precipitated out. This wasfiltered off, washed with water and methanol, then dried under vacuumover phosphorous pentoxide to give 948 mg (100%) of2,9-dimethoxyquinacridone.

Mass spectrum (ES+) (M+H) 373.

10.4 Diethyl ester of6-{2,9-dimethoxy-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid.

2,9-Dimethoxyquinacridone (375 mg; 1.0 mmol) was suspended in a mixtureof anhydrous dimethylformamide (5 ml) and anhydrous dimethyl sulphoxide(5 ml) under a nitrogen atmosphere. Sodium hydride (60% suspension inoil; 100 mg; 2.40 mmol) was added and the mixture stirred untileffervescence stopped. The reaction was heated to 70° C. for 1 hour.Ethyl 6-bromohexanoate (535 μl; 3.0 mmol) was added to the dark greensolution and the mixture stirred overnight at 70° C. The dark purple-redsolution was allowed to cool, then the mixture was poured into water(150 ml) and 1.0M hydrochloric acid (20 ml). The solid was filtered off,washed with water and air dried. This was purified by flashchromatography (silica. 5-30% ethyl acetate/dichloromethane) to give 230mg (35%) of6-{2,9-dimethoxy-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester as a red solid. δ_(H) (200 MHz, CDCl₃) 1.25(6H, t),1.80(12H, m), 2.40(4H, t), 4.00(6H, s), 4.15(4H, m), 4.50(4H, t ),7.42(4H, m), 7.91(2H, d), 8.70(2H, s). λ_(max)(ab) 510 nm, 547 nm.λ_(max)(em) 592 nm. (methanol).

Mass spectrum (ES+) (M+H) 656 (M+Na) 679.

11.6-{2,9-Dinitro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester

5,12-Bis(O-ethyl-6-hexanoyl)-5,12-dihydroquino[2,3-b]acridin-7,14-dione(300 mg; 0.5 mmol) was cooled in an ice bath and then dissolved in conc.sulphuric acid (3 ml) under a nitrogen atmosphere to give a purplesolution. Conc. nitric acid (70 μl; 1.08 mmol) was added and thereaction mix removed from the ice bath. After one hour, the reaction mixwas added to ice when an orange precipitate formed. The mixture wasextracted with dichloromethane. The organic phase was washed with dilutesodium bicarbonate solution, then dried with anhydrous magnesiumsulphate. After filtration, the solvent was removed by rotaryevaporation to give an orange solid. This was purified by flashchromatography (silica. 2-3% methanol/dichloromethane). After removal ofsolvent, the residue was triturated with diethyl ether to give 240 mg(70%) of6-{2,9-dinitro-12-(5-carboxypentyl)-7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid, diethyl ester as an orange solid.

δ_(H) (200 MHz, CD₃OD) 1.28(6H, t), 1.80(12H, m), 2.43(4H, t), 4.17(4H,dd), 4.56(4H, t), 7.56(2H, d), 8.48(2H, dd), 8.66(2H, s), 9.30(2H, d).λ_(max)(ab) 408 nm, 474 nm, 506 nm. λ_(max)(em) 518 nm, 556 nm.(Dichloromethane).

N.B. This material has very weak or is non-fluorescent in DMF, DMSO andmethanol.

12. Fluorescence Lifetime Studies

FIG. 2 is a plot showing the fluorescence lifetimes of certain dyesaccording to the invention. Fluorescence lifetimes of a range of dyeswere determined by a time-correlated single photon counting techniqueusing an Edinburgh Instruments FL900 CDT Time-Resolved Fluorometer.Samples were excited at 500 nm using a hydrogen-filled flashlamp.Detection was at 550 nm. Deconvolution using a non-linear least-squaresalgorithm gave the results shown in Table 2.

TABLE 2 Fluorescence Lifetimes Compound Solvent Lifetime5,12-Di-n-hexylquinacridone CH₂Cl₂/MeOH 26.5 nsec 2,4,9,11-Quinacridonetetrasulphonic acid water 22.1 nsec 2,9-Quinacridone disulphonic acidwater 20.6 nsec 6-(7,14-Dioxo-2,9-disulpho-7,14-dihydro- water 20.1 nsec12H-quino[2,3-b]acridin-5-yl)hexanoic acid Quinacridone DMSO   22 nsec2-Bromo-5,12-di-n-hexylquinacridone CH₂Cl₂/MeOH 20.4 nsec2,9-Dibromo-5,12-di-n-hexylquinacridone CH₂Cl₂/MeOH 16.9 nsec6-(12-Ethyl-7,14-dioxo-2,9-disulpho-7,14- water 22.7 nsecdihydro-12H-quino[2,3-b]acridin-5-yl) hexanoic acid6-{2,9-Dibromo-12-(5-carboxypentyl)-7,14- water 18.0 nsecdioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 5-yl}hexanoic acid, diethylester. 6-{2,9-Dibromo-12-(5-carboxypentyl)-7,14- MeOH/water 20.7 nsecdioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 17.7 nsec 5-yl}hexanoicacid. 6-{2,9-Dichloro-12-(5-carboxypentyl)-7,14- MeOH 22.3 nsecdioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 5-yl}hexanoic acid, diethylester. 6-{2,9-Difluoro-12-(5-carboxypentyl)-7,14- MeOH 21.4 nsecdioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 5-yl}hexanoic acid, diethylester. 6-{2,9-Dimethyl-12-(5-carboxypentyl)-7,14- MeOH 21.9 nsecdioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 5-yl}hexanoic acid, diethylester 6-{2,9-Dimethoxy-12-(5-carboxypentyl)- EtOH 14.0 nsec7,14-dioxo-7,14-dihydro-12H-quino[2,3-b]- acridin-5-yl}hexanoic acid,diethyl ester. 6-{2,9-Dinitro-12-(5-carboxypentyl)-7,14- CH₂Cl₂ 17.0nsec dioxo-7,14-dihydro-12H-quino[2,3-b]acridin- 5-yl}hexanoic acid,diethyl ester.

13. Protein Labelling

13.1 Preparation of a conjugate of6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid with ovalbumin

To 10 ml of ovalbumin (1 mg/ml in 0.1M carbonate buffer, pH 9.3) wasadded 100 μl ofO—(N-succinimidyl)-6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid (1 mg/100 μl in DMSO) dropwise whilst stirring. Gentle stirringcontinued for 1 hr at ambient temperature in a foil wrapped vial.Unconjugated dye was removed by overnight dialysis (12-14K MWCO) at +4°C. with at least 2 changes of PBS. The dye-conjugate (Conjugate A) wasrecovered and stored at +4° C.

13.2 Determination of the Fluorescence Lifetimes of6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid and its conjugate with ovalbumin (Conjugate A)

The fluorescence lifetimes of6-{7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid and its conjugate with ovalbumin (Conjugate A) was determined inPBS. The results are shown in FIG. 3. Deconvolution and curve fittingusing a non-linear least-squares algorithm gave the results shown inTable 3.

TABLE 3 Fluorescence Lifetimes Name Lifetime (nsec)6-{7,14-Dioxo-2,9-disulpho-7,14-dihydro-12H- 20.1quino[2,3-b]acridin-5-yl}hexanoic acid6-{7,14-Dioxo-2,9-disulpho-7,14-dihydro-12H- 19.8quino[2,3-b]acridin-5-yl}hexanoic acid - Ovalbumin conjugate (ConjugateA)

14. Labelling of MMP3 peptide substrate withO—{N-succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate

The MMP3 peptide substrate (NH₂-RPKPVE(Nva)WRK-NH₂) was synthesised on aApplied Biosystems model 431A peptide synthesiser using standard Fmocchemistry and Rink amide resin. At the end of the synthesis theN-terminal Fmoc group was removed. The partially protected peptide wasleft attached to the solid support, in which form it was reacted withO—{N-succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate.The labelled peptide was then cleaved from the solid support usingstandard techniques and then purified by reverse phase HPLC.

50 mg of the resin bound peptide (equivalent to 16 μmoles of peptide)was weighed into a 1.5 ml screw top polypropylene V-vial to which wasadded 12 mg (16 μmoles) ofO-{N-succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid dissolved in 1 ml of anhydrous DMSO followed by 20 μl ofdiisopropylethylamine. The vial was placed on rollers with lightexcluded for 20 hrs at ambient temperature (22° C.). The resin was thenfiltered off using a sintered glass frit, washed with 5 ml dry DMSO, 5ml methanol and finally 5 ml dichloromethane, then dried in vacuo for 2hrs.

The resin was placed in a small round bottomed flask to which was added2 ml of an ice cold solution of trifluoroacetic acid (1.9 ml), water (50l) and triisopropylsilane (TIS)(50 μl). The mixture was stirredmagnetically for 90 minutes and allowed to warm to ambient temperature.The mixture was then filtered through a glass wool plug and allowed todrip into 10 ml of ice cold diethyl ether. The pale yellow precipitatewas spun down, the supernatant removed, the precipitate redissolved in 1ml trifluoroacetic acid and reprecipitated in 10 ml ice cold ether. Theprecipitate was spun down, washed twice with ether then dried in vacuo.

The crude labelled peptide was dissolved in water, filtered through a0.45 um Millipore filter and a portion was purified on a 25 cm×1 cm C-18Phenomenex Jupiter column (code 00G-4055-N0) using a gradient of 0.1%TFA/water to 100% of 0.1% TFA/acetonitrile over 30 minutes and a flow of4 ml/minute. Detection was at 220 and 500 nm. One major peaks was elutedafter 13 minutes. The material was freeze dried to give 11.4 mg (6.0 μm)of a red solid.

Mass spectrum (ES+) (M+H) 1893 (calculated molecular weight ofquinacridone labelled peptide=1892).

15. Trypsin Cleavage of a Conjugate of Albumin with6-{12-Ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoicacid Monitored by Fluorescence Polarisation 15.1 Preparation of AlbuminConjugate

To 10 ml of human serum albumin (2 mg/ml in 0.1M carbonate buffer,pH9.3), was addedO—{N-succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl}hexanoate(110 μl; 2 mg/ml in DMSO) dropwise whilst stirring. Gentle stirringcontinued for 1 hr at ambient temperature in a foil wrapped vial. ASephadex G25 column (PD10—Amersham Biosciences) was used to purify theconjugate which was eluted in de-ionised water.

15.2 Trypsin Cleavage of Albumin Conjugate

To 10 μl of the conjugate (20 μg) in 2 ml of buffer (20 mM Tris pH 7.5;200 mM NaCl; 6 mM CaCl₂) in a cuvette, either 50 μl buffer (as no enzymecontrol) or 50 μl (500 μg) of trypsin was added. Measurement of thefluorescence polarisation signal was performed using a FluoroMax-3spectrofluorometer (JYHoriba), with excitation at 485 nm, and detectionat 555 nm for 20 minutes at ambient temperature. The results asillustrated in FIG. 4, show that the signal becomes less polarised asthe albumin conjugate is cleaved into smaller fragments by trypsin, asexpected from polarisation theory.

1. A compound having the formula:

wherein: groups R³ and R⁴ are attached to the Z¹ ring structure andgroups R⁵ and R⁶ are attached to the Z² ring structure; Z¹ and Z²independently represent the atoms necessary to complete a phenyl ringstructure; at least one of groups R¹ and R² is the group -E-F, wherein Eis a spacer group selected from the group consisting of: (CHR′)_(p)——{(CHR′)_(q)—O—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—NR′—(CHR′)_(r)}_(s)——{(CHR′)_(q)—(CH═CH)—(CHR′)_(r)}_(s)— —{(CHR′)_(q)—Ar—(CHR′)_(r)}_(s)——{(CHR′)_(q)—CO—NR′—(CHR′)_(r)}_(s)— and—{(CHR′)_(q)—CO—Ar—NR′—(CHR′)_(r)}_(s)— where R′ is hydrogen, C₁-C₄alkyl or aryl, which may be optionally substituted with sulphonate, Aris phenylene, optionally substituted with sulphonate, p is 1-10, q is0-10, r is 1-10 and s is 1-5; and F is a reactive group selected fromthe group consisting of carboxyl, succinimidyl ester,sulpho-succinimidyl ester, isothiocyanate, maleimide, haloacetamide,acid halide, hydrazide, vinylsulphone, dichlorotriazine andphosphoramidite; groups R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independentlyselected from hydrogen, halogen, amide, hydroxyl, cyano, nitro, C₁-C₆alkoxy, C₁-C₂₀ alkyl, sulphonate, and sulphonic acid; when any of groupsR¹ and R² is not said group -E-F, said remaining group R¹ or R² isselected from hydrogen, mono- or di-nitro-substituted benzyl, C₁-C₂₀alkyl, and the group —(CH₂—)_(n) Y; Y is selected from sulphonate,sulphate, phosphonate, phosphate, quaternary ammonium and carboxyl; andn is an integer from 1 to
 6. 2. The compound of claim 1, wherein saidcompound is a fluorescent dye and further wherein: groups R³, R⁴, R⁵,R⁶, R⁷ and R⁸ are independently selected from hydrogen, halogen, amide,hydroxyl, cyano, C₁-C₆ alkoxy, acrylate, vinyl, styryl, aryl,heteroaryl, C₁-C₂₀ alkyl, sulphonate, and sulphonic acid; and remaininggroups R¹ and R² are independently selected from hydrogen, C₁-C₂₀ alkyl,and the group —(CH₂—)_(n)Y.
 3. The compound of claim 1, wherein saidcompound is a non-fluorescent or substantially non-fluorescent dye andfurther wherein at least one of groups R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸comprises at least one nitro group.
 4. The compound of claim 1, whereingroup -E-F is selected from:

wherein n is an integer from 1-10.
 5. The compound of claim 4, wherein nis
 5. 6. A compound selected from: i)O—(N-succinimidyl)-6-(7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3 -b]acridin-5-yl)hexanoate; and ii)O—{N-succinimidyl-6-(12-ethyl-7,14-dioxo-2,9-disulpho-7,14-dihydro-12H-quino[2,3-b]acridin-5-yl)}hexanoate.